Project Summary The NLRP3 inflammasome has been linked to both protective and pathologic immune responses. Its appropriate activation triggers the innate immune response to invading pathogens including influenza virus, Staphylococcus aureus and Candida albicans; in contrast aberrant activation of the NLRP3 inflammasome is implicated in the pathogenesis of a variety of sterile inflammatory diseases. Activation of the NLRP3 inflammasome is a multistep process that culminates in the activation of the cysteine protease caspase-1, which subsequently results in the processing and secretion of the proinflammatory cytokines IL-1? and IL-18. The precise steps leading to activation of the NLRP3 inflammasome are not well understood but involve mitochondrial dysfunction and cation flux. Importantly we have found that the mitochondrial specific lipid cardiolipin directly binds to NLRP3 and caspase-1 and is required for NLRP3 inflammasome activation. We propose that the mitochondrion serves as an activating platform for the assembly and activation of the NLRP3 inflammasome. We will define how the mitochondrion specifically interacts with NLRP3 inflammasome components during the priming and activation steps in NLRP3 inflammasome. We will determine if calcium flux, the generation of reactive oxygen species, and changes in mitochondrial dynamics trigger the critical interaction between NLRP3 inflammasome components and the mitochondrion. Numerous sterile inflammatory processes are caused by inappropriate activation of the NLRP3 inflammasome. We have previously shown the inflammatory response to acetaminophen-induced hepatotoxicity and pulmonary silicosis is dependent upon activation of the NLRP3 inflammasome. We will use these two clinically relevant models of NLRP3-dependent pathogenic sterile inflammation to identify novel therapeutic targets that can disrupt activation of the NLRP3 inflammasome in vivo.